DNA encoding human vanilloid receptor VR3

ABSTRACT

DNA encoding human VR1 receptor has been cloned and characterized. The recombinant protein is capable of forming biologically active protein. The cDNA&#39;s have been expressed in recombinant host cells that produce active recombinant protein. The recombinant protein is also purified from the recombinant host cells. In addition, the recombinant host cells are utilized to establish a method for identifying modulators of the receptor activity, and receptor modulators are identified.

BACKGROUND OF THE INVENTION

Noxious chemical, thermal and mechanical stimuli excite peripheral nerveendings of small diameter sensory neurons (nociceptors) in sensoryganglia (e.g., dorsal root, nodose and trigeminal ganglia) and initiatesignals that are perceived as pain. These neurons are crucial for thedetection of harmful or potentially harmful stimuli (heat) and tissuedamage (H⁺ (local tissue acidosis), and/or stretch) which arise fromchanges in the extracellular space during inflammatory or ischaemicconditions (Wall and Melzack, 1994). The vanilloid capsaicin(8-methyl-N-vanillyl-6-nonenamide), the main pungent ingredient in “hot”capsicum peppers, is a very selective activator of thinly orunmyelinated nociceptive afferents (Szolcsanyi, 1993; Szolcsanyi, 1996).Electrophysiological studies have shown that vanilloids excite smallsensory neurons by activating a plasma membrane channel that isnon-selectively permeable to cations (Bevan and Szolcsanyi, 1990; Oh etal., 1996; Wood et al., 1988). The ultra potent tricyclic diterpeneresiniferatoxin from Euphorbia plants (RTX; (Szolcsanyi et al., 1991))binds with nanomolar affinity at the capsaicin binding site and hasrevealed a very localized distribution of capsaicin receptors to ratsomatic and visceral primary sensory neurons (Szallasi, 1995).

The vanilloid receptor VR1 (Caterina et al., 1997) is thought to be aheat-sensing receptor whose threshold is decreased in the presence ofprotons or capsaicin (Tominaga et al., 1998). Capsaicin and protonsinteract at specific membrane recognition sites (vanilloid receptors)expressed almost exclusively by primary sensory neurons involved innociception and neurogenic inflammation (Bevan and Szolcsanyi, 1990).The vanilloid (“capsaicin”) receptor VR1 is activated by capsaicin andRTX, and activation of VR1 is blocked by the antagonists capsazepine(CPZ; (Bevan et al., 1992)) and ruthenium red (RR; (Caterina et al.,1997; Wood et al., 1988)).

Hydropathicity analysis of the amino acid sequence of VR1 reveals 6potential membrane spanning regions (S1-S6) and a putative pore-loopregion between S5 and S6. A large intracellular domain contains 3ankyrin repeat domains. (Caterina et al., 1997). This channel hassignificant structural similarities with the putative “store-operated”TRP calcium channel family. VR1 is a ligand-gated non-selective cationchannel that shows pronounced outward rectification (Caterina et al.,1997). Importantly, VR1 is highly permeable to Ca2+, an ion known to bevery important in regulating cell function ((Blackstone and Sheng, 1999;Gupta and Pushkala, 1999; van Haasteren et al., 1999)).

Searching genomic databases has revealed VRL-1, a subunit structurallyrelated to VR1. Rat and human VRL-1 (AF129113 and AF129112,respectively) are ˜49% identical and 66% similar to rat VR1 (AF029310))(Caterina et al., 1999). Human VRL protein (AF103906) cloned by Wood andcollegues (unpublished) is 99% identical to VRL-1 (AF129112). Recently,a patent application by Partiseti and Renard was published thatdescribed hVRCC (human vanilloid receptor like cation channel) which isnearly identical to AF129112 (the only difference is the deletion ofQ418). We will refer to these sequences as VR2. Overall, the predictedstructure of VR1 and VR2 is characteristic of a family of ion channelsdefined by the transient receptor potential (TRP) channels originallycloned from Drosophila melanogaster, a Ca-permeable channel that plays arole in phototransduction (Lu and Wong, 1987; Minke and Selinger, 1996).This receptor appears to also be involved in the sensation ofpain-producing heat (Caterina et al., 1999). Expression of VR2 inoocytes and HEK cells usually conferred a sensitivity of the cells tonoxious temperatures (>53 degC.), that was not sensitive to CPZ but wasnearly completely blocked at 10 μM ruthenium red. Activation of VR2induces a non-selective cation current with high permeability to Ca2+.Interestingly, the threshold for heat sensitivity decreased withrepeated application of noxious stimuli, but not subthresholdtemperatures (Caterina et al., 1999).

The rat SIC (stretch-inhibitable channel; Genbank AB015231), encoded by529 amino acids, is thought to form an ion channel inhibited by stretch(Suzuki et al., 1999). The first 379 amino acids homologous to rat VR1.SIC lacks the large N-terminal cytoplasmic domain of the VR family butcontains a sequence homologous to the A exon prior to the putative TM1.The last 163 amino acids, beginning in the middle of putative TM6 of ratSIC are similar to the corresponding amino acid sequence of the humanVR3 A+B− of the present invention.

The present invention describes the cloning and function of a novelvanilloid receptor family member, VR3. This gene appears to bealternatively spliced to create at least 3 isoforms.

SUMMARY OF THE INVENTION

DNA molecules encoding 3 isoforms of the human vanilloid receptor 3(hVR3) have been cloned and characterized. The biological and structuralproperties of these proteins are disclosed, as is the amino acid andnucleotide sequence. The recombinant protein is useful to identifymodulators of the receptor VR3. Modulators identified in the assaydisclosed herein are useful as therapeutic agents, which are candidatesfor the treatment of inflammatory conditions and for use as analgesicsfor intractable pain associated with postherpetic neuralgia, diabeticneuropathy, postmastectomy pain, complex regional pain syndromes,arthritis (e.g., rheumatoid and osteoarthritis), as well as ulcers,neurodegenerative diseases, asthma, chronic obstructive pulmonarydisease, irritable bowel syndrome, and psoriasis. Uses include thetreatment of central nervous system diseases, diseases of the intestinaltract, abnormal proliferation and cancer especially in the digestivesystem, prostate and female gonads, ulcer, liver disease, kidneydisease, control of viscera innervated by the dorsal root ganglia, or todiagnose or treat any disorder related to abnormal expression of thesehVR3 polypeptides, among others. In another aspect, the inventionrelates to methods to identify agonists and antagonists using thematerials provided by the invention, and treating conditions associatedwith hVR3 imbalance. The recombinant DNA molecules, and portionsthereof, are useful for isolating homologues of the DNA molecules,identifying and isolating genomic equivalents of the DNA molecules, andidentifying, detecting or isolating mutant forms of the DNA molecules.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A and 1B—SEQ.ID.NO.:5. Human VR3A+B− nucleotide sequence of thecoding region (2616 bp).

FIGS. 2A and 2B—SEQ.ID.NO.:6. The nucleotide sequence of human VR3A+B−is shown including 337 bp 5′ untranslated region (UT) and 547 bp 3′UT(3500 bp).

FIG. 3—SEQ.ID.NO.:7. Coding sequence for human VR3A+B− (871 amino acids)

FIGS. 4A and 4B—SEQ.ID.NO.:8. Human VR3A−B− nucleotide sequence of thecoding region (2436 bp).

FIG. 5—SEQ.ID.NO.:9. Coding sequence for human VR3A−B− (811 amino acids)

FIG. 6—SEQ.ID.NO.:10. Human VR3A+B+ nucleotide sequence of the codingregion (2229 bp).

FIGS. 7A AND 7B—SEQ.ID.NO.:11. The nucleotide sequence of human VR3A+B+is shown including 836 bp 5′ UT and 994 bp 3′UT (4059 bp).

FIG. 8—SEQ.ID.NO.:12. Coding sequence for human VR3A+B+ (742 aminoacids)

FIG. 9—Functional expression of VR3 isoforms in Xenopus oocytes isshown: viability of oocyte maintained in ND-96 with 2 Ca²⁺wassignificantly diminished 4-6 days after injection with 3.25 ng VR3 A+B−and VR3 A+B+ (5 ng) but not VR3 A−B− (1.5 ng) cRNA. Data for VR3 A+B−and water-injected oocytes were obtained from 5 different experiments.Dead oocytes were determined visually. Data were analyzed using Chisquare analysis.

FIGS. 10A AND 10B—Function in oocytes: VR3 isoforms are activated byheat. FIG. 10A. Shown is the mean peak current elicited by a heat rampfrom 25 deg C. to 46 deg C. and maintained at 46 degC. for at least 15sec. The current is the increase over initial current at +80 mV. Voltageramps were applied from −120 to +80 mV over 400 msec every 2 sec.Oocytes were injected with 3.25, 1.5 and 5 ng VR3 A+B−, A−B−, and A+B+cRNA, respectively, and recorded up to 6 days later. Solid bar: waterinjected controls (n=8); clear bar: VR3 A+B− isoform (n=11); hatchedbar: VR3 A−B− isoform (n=8); stippled bar: VR3 A+B+ isoform (n=5). Allisoforms show a significant increase over water controls (p=0.001, 0.03and 0.007, respectively; Student's t-test). The heat induced response isabout 2-fold larger in the A+B− isoform compared to the other 2 isoformsbut the differences are not significant (p=0.051 and p=0.16, for A+B−compared to A−B− and A+B+, respectively). Data were obtained from 2 setsof injected oocytes. Data shown is the mean and standard error of themean. FIG. 10B. Voltage-ramp induced currents were recorded duringapplication of increasing heat to oocytes injected with water (top), VR3A+B− (second from top), VR3 A−B− (3^(rd) from top), and VR3A+B+(bottom). Oocytes were constantly perfused with Ca2+ ND-96 and thesolution was heated by an inline heater device (TC-324B in conjunctionwith the SH-27A in line heater; Warner Instrument Corp.). Ramp inducedcurrents (in uA, as indicated on the y-axis) obtained at temperaturesfrom 37 deg C. to 46 deg C. are displayed; only current traces at thehigher temperatures are labeled with the corresponding temperature.

FIGS. 11A and 11B—Function in oocytes: VR3A+B− confers sensitivity to 10uM ruthenium red to the perfusion activated current (I_(perfusion))observed in VR1-expressing oocytes induced by cell perfusion byextracellular saline solutions. Activation of I_(perfusion) by increasedperfusion was blocked by ruthenium red only in VR1- expressing oocytesinjected with VR3 A+B− cRNA, and not in oocytes expressing only VR1.FIG. 11A. An oocyte injected with VR1 cRNA [4 ng] was challenged withvoltage ramps between −120 and +80 mV over 400 msec from a holdingpotential of −70 mV. The ramp-induced currents were increased afteronset of perfusion of the oocyte at a rate of 10 ml/min. Preincubationwith 10 uM ruthenium red for 0.5 min blocked the current. The block waspartially reversible (not shown). FIG. 11B. An oocyte injected with VR1cRNA [4 ng] together with VR3 A+B− [1.3 ng] was challenged with voltageramps between −120 and +80 mV over 400 msec from a holding potential of−70 mV. The ramp-induced currents were increased after onset ofperfusion of the oocyte at a rate of 10 ml/min. Preincubation with 10 uMruthenium red for 0.5 min had little effect on I_(perfusion).I_(perfusion) had similar magnitudes in both sets of oocytes. Vrev forthe RR inhibited current was about −13 mV (arrow).

FIGS. 12A and 12B—Function in oocytes: FIG. 12A—VR3A+B− together withVR1. FIG. 12B—VR1 alone. The magnitude and decay kinetics of theresponse to 1 uM capsaicin was diminished when VR1 cRNA was co-expressedwith VR3 A+B− cRNA at equal ratios [2.9 ng each].

FIG. 13—DNA array distribution analysis indicates that hVR3 mRNAs areexpressed in a variety of tissues, and there is some overlap ofexpression with VR1 at a whole tissue level. The DNA sequence used onthe DNA array is not present in A+B+, only A+B− and A−B− isoforms. Notethat the cDNA species was cloned from pituitary and prostate glands.

DETAILED DESCRIPTION

The present invention describes 3 isoforms of a human vanilloidreceptor, termed VR3: VR3A+B−, VR3A−B−, and VR3A+B+. The nucleotidesequences of human VR3 receptor cDNAs revealed single large open readingframe of about 2616 (FIGS. 1A and 1B), 2436 (FIGS. 4A and 4B) and 2229(FIG. 6) base pairs encoding 871 (FIG. 3), 811 (FIG. 5), and 742 (FIG.8) amino acids for human VR3 A+B−, A−B− and A+B+, respectively. The cDNAfor VR3 A+B− has 5′ and 3′-untranslated extensions of about 337 andabout 547 nucleotides, as shown in FIGS. 2A and 2B, wherein the 5′UTR is1-337, the coding region is 338-2953, and the 3′UTR is 2954-3500. ThecDNA for VR3 A+B+ has 5′ and 3′-untranslated extensions of about 836 andabout 994 nucleotides as shown in FIGS. 7A and 7B, wherein the 5′UTR is1-836, the coding region is 837-3065, and the 3′UTR is 3066-4059. Thefirst in-frame methionine was designated as the initiation codon for anopen reading frame that predicts human VR3 receptor proteins with anestimated molecular mass (M_(r)) of about 98,242 Da, 91,294 Da and83,310 Da for the isoforms A+B−, A−B− and A+B+, respectively. The A+B−isoform encodes a protein of 871 amino acids. The VR3 A−B− contains adeletion of 60 amino acids from amino acid 382 to 441 of VR3A+B−. VR3A+B+ is identical to A+B− until amino acid 736 after which there are 6divergent amino acids and a stop codon. The VR3 A+B+ isoform extends 20amino acids after the putative TM6.

The predicted human VR3 receptor proteins were aligned with nucleotideand protein databases and are related to the vanilloid receptor family(VR1 and VR2). There are several conserved motifs found in this familyof receptor including a large putative N-terminal hydrophilic segment(about 467 amino acids), three putative ankyrin repeat domains in theN-terminus region, 6 predicted transmembrane regions and a pore region.VR3 A+B− is 43% identical to human VR1, 39% identical to both humanVRL-1 (AF129112) and human VRL (AF103906). Thus the VR3 receptordescribed herein is clearly a novel gene of the vanilloid receptorfamily.

Human VR3A+B− and VR3A−B− forms are similar to the ratstretch-inhibitable channel SIC [Genbank accession ABO15231] from aminoacid 694 to the end. Rat SIC, encoded by 529 amino acids, is thought toform an ion channel inhibited by stretch. It lacks the large N-terminalcytoplasmic domain of the VR family but contains a sequence homologousto the A exon prior to the putative TM1.

The complete genomic sequence of the VR3 coding regions described hereinappears to be found in a 380512 base pair sequence submission to Genbank(homo sapiens clone RPCI1-7G5 (AC007834), direct submission by Worley,K. C.). This Genbank entry list many fragments of DNA sequence and aproposed contiguous sequence, but lacks any analysis of the nucleic acidsequence and fails to characterize the features of the VR3 nucleic acidsequences, or describe the presence of the VR3 gene

Isolation of Human VR3 Receptor Nucleic Acid

The present invention relates to DNA encoding human VR3 receptor whichwere isolated from human VR3 receptor producing cells. Human VR3receptor, as used herein, refers to protein which can specificallyfunction as a human vanilloid receptor.

The complete amino acid sequence of human VR3 receptor was notpreviously known, nor was the complete nucleotide sequence encodinghuman VR3 receptor known. It is predicted that a wide variety of cellsand cell types will contain the described human VR3 receptor.

Other cells and cell lines may also be suitable for use to isolate humanVR3 receptor cDNA. Selection of suitable cells may be done by screeningfor human VR3 receptor activity in cell extracts or in whole cellassays, described herein. Cells that possess human VR3 receptor activityin any one of these assays may be suitable for the isolation of humanVR3 receptor DNA or mRNA.

Any of a variety of procedures known in the art may be used tomolecularly clone human VR3 receptor DNA. These methods include, but arenot limited to, direct functional expression of the human VR3 receptorgenes following the construction of a human VR3 receptor-containing cDNAlibrary in an appropriate expression vector system. Another method is toscreen human VR3 receptor-containing cDNA library constructed in abacteriophage or plasmid shuttle vector with a labelled oligonucleotideprobe designed from the amino acid sequence of the human VR3 receptorsubunits. An additional method consists of screening a human VR3receptor-containing cDNA library constructed in a bacteriophage orplasmid shuttle vector with a partial cDNA encoding the human VR3receptor protein. This partial cDNA is obtained by the specific PCRamplification of human VR3 receptor DNA fragments through the design ofdegenerate oligonucleotide primers from the amino acid sequence of thepurified human VR3 receptor protein.

Another method is to isolate RNA from human VR3 receptor-producing cellsand translate the RNA into protein via an in vitro or an in vivotranslation system. The translation of the RNA into a peptide a proteinwill result in the production of at least a portion of the human VR3receptor protein which can be identified by, for example, immunologicalreactivity with an anti-human VR3 receptor antibody or by biologicalactivity of human VR3 receptor protein. In this method, pools of RNAisolated from human VR3 receptor-producing cells can be analyzed for thepresence of an RNA that encodes at least a portion of the human VR3receptor protein. Further fractionation of the RNA pool can be done topurify the human VR3 receptor RNA from non-human VR3 receptor RNA. Thepeptide or protein produced by this method may be analyzed to provideamino acid sequences which in turn are used to provide primers forproduction of human VR3 receptor cDNA, or the RNA used for translationcan be analyzed to provide nucleotide sequences encoding human VR3receptor and produce probes for this production of human VR3 receptorcDNA. This method is known in the art and can be found in, for example,Maniatis, T., Fritsch, E. F., Sambrook, J. in Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. 1989.

It is readily apparent to those skilled in the art that other types oflibraries, as well as libraries constructed from other cells or celltypes, may be useful for isolating human VR3 receptor-encoding DNA.Other types of libraries include, but are not limited to, cDNA librariesderived from other cells, from organisms other than human VR3 receptor,and genomic DNA libraries that include YAC (yeast artificial chromosome)and cosmid libraries.

It is readily apparent to those skilled in the art that suitable cDNAlibraries may be prepared from cells or cell lines which have human VR3receptor activity. The selection of cells or cell lines for use inpreparing a cDNA library to isolate human VR3 receptor cDNA may be doneby first measuring cell associated human VR3 receptor activity using themeasurement of human VR3 receptor-associated biological activity or aligand binding assay.

Preparation of CDNA libraries can be performed by standard techniqueswell known in the art. Well known cDNA library construction techniquescan be found for example, in Maniatis, T., Fritsch, E. F., Sambrook, J.,Molecular Cloning: A Laboratory Manual, Second Edition (Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989).

It is also readily apparent to those skilled in the art that DNAencoding human VR3 receptor may also be isolated from a suitable genomicDNA library. Construction of genomic DNA libraries can be performed bystandard techniques well known in the art. Well known genomic DNAlibrary construction techniques can be found in Maniatis, T., Fritsch,E. F., Sambrook, J. in Molecular Cloning: A Laboratory Manual, SecondEdition (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).

In order to clone the human VR3 receptor gene by the above methods, theamino acid sequence of human VR3 receptor may be necessary. Toaccomplish this, human VR3 receptor protein may be purified and partialamino acid sequence determined by automated sequenators. It is notnecessary to determine the entire amino acid sequence, but the linearsequence of two regions of 6 to 8 amino acids from the protein isdetermined for the production of primers for PCR amplification of apartial human VR3 receptor DNA fragment.

Once suitable amino acid sequences have been identified, the DNAsequences capable of encoding them are synthesized. Because the geneticcode is degenerate, more than one codon may be used to encode aparticular amino acid, and therefore, the amino acid sequence can beencoded by any of a set of similar DNA oligonucleotides. Only one memberof the set will be identical to the human VR3 receptor sequence but willbe capable of hybridizing to human VR3 receptor DNA even in the presenceof DNA oligonucleotides with mismatches. The mismatched DNAoligonucleotides may still sufficiently hybridize to the human VR3receptor DNA to permit identification and isolation of human VR3receptor encoding DNA. DNA isolated by these methods can be used toscreen DNA libraries from a variety of cell types, from invertebrate andvertebrate sources, and to isolate homologous genes.

Purified biologically active human VR3 receptor may have severaldifferent physical forms. human VR3 receptor may exist as a full-lengthnascent or unprocessed polypeptide, or as partially processedpolypeptides or combinations of processed polypeptides. The full-lengthnascent human VR3 receptor polypeptide may be posttranslationallymodified by specific proteolytic cleavage events that results in theformation of fragments of the full length nascent polypeptide. Afragment, or physical association of fragments may have the fullbiological activity associated with human VR3 receptor however, thedegree of human VR3 receptor activity may vary between individual humanVR3 receptor fragments and physically associated human VR3 receptorpolypeptide fragments.

Because the genetic code is degenerate, more than one codon may be usedto encode a particular amino acid, and therefore, the amino acidsequence can be encoded by any of a set of similar DNA oligonucleotides.Only one member of the set will be identical to the human VR3 receptorsequence but will be capable of hybridizing to human VR3 receptor DNAeven in the presence of DNA oligonucleotides with mismatches underappropriate conditions. Under alternate conditions, the mismatched DNAoligonucleotides may still hybridize to the human VR3 receptor DNA topermit identification and isolation of human VR3 receptor encoding DNA.

DNA encoding human VR3 receptor from a particular organism may be usedto isolate and purify homologues of human VR3 receptor from otherorganisms. To accomplish this, the first human VR3 receptor DNA may bemixed with a sample containing DNA encoding homologues of human VR3receptor under appropriate hybridization conditions. The hybridized DNAcomplex may be isolated and the DNA encoding the homologous DNA may bepurified therefrom.

It is known that there is a substantial amount of redundancy in thevarious codons that code for specific amino acids. Therefore, thisinvention is also directed to those DNA sequences that containalternative codons that code for the eventual translation of theidentical amino acid. For purposes of this specification, a sequencebearing one or more replaced codons will be defined as a degeneratevariation. Also included within the scope of this invention aremutations either in the DNA sequence or the translated protein that donot substantially alter the ultimate physical properties of theexpressed protein. For example, substitution of valine for leucine,arginine for lysine, or asparagine for glutamine may not cause a changein functionality of the polypeptide. Such substitutions are well knownand are described, for instance in Molecular Biology of the Gene, 4^(th)Ed. Benjamin Cummings Pub. Co. by Watson et al.

It is known that DNA sequences coding for a peptide may be altered so asto code for a peptide having properties that are different than those ofthe naturally occurring peptide. Methods of altering the DNA sequencesinclude, but are not limited to site directed mutagenesis, chimericsubstitution, and gene fusions. Site-directed mutagenesis is used tochange one or more DNA residues that may result in a silent mutation, aconservative mutation, or a nonconservative mutation. Chimeric genes areprepared by swapping domains of similar or different genes to replacesimilar domains in the human VR3 receptor gene. Similarly, fusion genesmay be prepared that add domains to the human VR3 receptor gene, such asan affinity tag to facilitate identification and isolation of the gene.Fusion genes may be prepared to replace regions of the human VR3receptor gene, for example to create a soluble version of the protein byremoving a transmembrane domain or adding a targeting sequence toredirect the normal transport of the protein, or adding newpost-translational modification sequences to the human VR3 receptorgene. Examples of altered properties include but are not limited tochanges in the affinity of an enzyme for a substrate or a receptor for aligand.

As used herein, a “functional derivative” of human VR3 receptor is acompound that possesses a biological activity (either functional orstructural) that is substantially similar to the biological activity ofhuman VR3 receptor. The term “functional derivatives” is intended toinclude the “fragments,” “variants,” “degenerate variants,” “analogs”and “homologues” or to “chemical derivatives” of human VR3 receptor. Theterm “fragment” is meant to refer to any polypeptide subset of human VR3receptor. The term “variant” is meant to refer to a moleculesubstantially similar in structure and function to either the entirehuman VR3 receptor molecule or to a fragment thereof. A molecule is“substantially similar” to human VR3 receptor if both molecules havesubstantially similar structures or if both molecules possess similarbiological activity. Therefore, if the two molecules possesssubstantially similar activity, they are considered to be variants evenif the structure of one of the molecules is not found in the other oreven if the two amino acid sequences are not identical. The term“analog” refers to a molecule substantially similar in function toeither the entire human VR3 receptor molecule or to a fragment thereof.

Recombinant Expression of Human VR3 Receptor

The cloned human VR3 receptor DNA obtained through the methods describedherein may be recombinantly expressed by molecular cloning into anexpression vector containing a suitable promoter and other appropriatetranscription regulatory elements, and transferred into prokaryotic oreukaryotic host cells to produce recombinant human VR3 receptor protein.Techniques for such manipulations are fully described in Maniatis, T, etal., supra, and are well known in the art.

Expression vectors are defined herein as DNA sequences that are requiredfor the transcription of cloned copies of genes and the translation oftheir mRNAs in an appropriate host. Such vectors can be used to expresseukaryotic genes in a variety of hosts such as bacteria including E.coli, blue-green algae, plant cells, insect cells, fungal cellsincluding yeast cells, and animal cells.

Specifically designed vectors allow the shuttling of DNA between hostssuch as bacteria-yeast or bacteria-animal cells or bacteria-fungal cellsor bacteria-invertebrate cells. An appropriately constructed expressionvector should contain: an origin of replication for autonomousreplication in host cells, selectable markers, a limited number ofuseful restriction enzyme sites, a potential for high copy number, andactive promoters. A promoter is defined as a DNA sequence that directsRNA polymerase to bind to DNA and initiate RNA synthesis. A strongpromoter is one that causes mRNAs to be initiated at high frequency.Expression vectors may include, but are not limited to, cloning vectors,modified cloning vectors, specifically designed plasmids or viruses.

A variety of mammalian expression vectors may be used to expressrecombinant Human VR3 receptor in mammalian cells. Commerciallyavailable mammalian expression vectors which may be suitable forrecombinant Human VR3 receptor expression, include but are not limitedto, pMAMneo (Clontech), pcDNA3 (InVitrogen), pMC1neo (Stratagene), pXT1(Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2)(ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199),pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and1ZD35 (ATCC 37565).

A variety of bacterial expression vectors may be used to expressrecombinant human VR3 receptor in bacterial cells. Commerciallyavailable bacterial expression vectors which may be suitable forrecombinant human VR3 receptor expression include, but are not limitedto pET vectors (Novagen) and pQE vectors (Qiagen).

A variety of fungal cell expression vectors may be used to expressrecombinant human VR3 receptor in fungal cells such as yeast.Commercially available fungal cell expression vectors which may besuitable for recombinant human VR3 receptor expression include but arenot limited to pYES2 (InVitrogen) and Pichia expression vector(InVitrogen). A variety of insect cell expression vectors may be used toexpress recombinant human VR3 receptor in insect cells. Commerciallyavailable insect cell expression vectors which may be suitable forrecombinant expression of human VR3 receptor include but are not limitedto pBlueBacII (InVitrogen).

DNA encoding human VR3 receptor may be cloned into an expression vectorfor expression in a recombinant host cell. Recombinant host cells may beprokaryotic or eukaryotic, including but not limited to bacteria such asE. coli fungal cells such as yeast, mammalian cells including but notlimited to cell lines of human, bovine, porcine, monkey and rodentorigin, and insect cells including but not limited to drosophila andsilkworm derived cell line. Cell lines derived from mammalian specieswhich may be suitable and which are commercially available, include butare not limited to, CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7(ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCCCRL 1658), HeLa (ATCC CCL 2), C1271 (ATCC CRL 1616), BS-C-1 (ATCC CCL26), MRC-5 (ATCC CCL 171), L-cells, and HEK-293 (ATCC CRL1573).

The expression vector may be introduced into host cells via any one of anumber of techniques including but not limited to transformation,transfection, protoplast fusion, lipofection, and electroporation. Theexpression vector-containing cells are clonally propagated andindividually analyzed to determine whether they produce human VR3receptor protein. Identification of human VR3 receptor expressing hostcell clones may be done by several means, including but not limited toimmunological reactivity with anti-human VR3 receptor antibodies, andthe presence of host cell-associated human VR3 receptor activity.

Expression of human VR3 receptor DNA may also be performed using invitro produced synthetic mRNA. Synthetic mRNA or mRNA isolated fromHuman VR3 receptor producing cells can be efficiently translated invarious cell-free systems, including but not limited to wheat germextracts and reticulocyte extracts, as well as efficiently translated incell based systems, including but not limited to microinjection intofrog oocytes, with microinjection into frog oocytes being generallypreferred.

To determine the human VR3 receptor DNA sequence(s) that yields optimallevels of human VR3 receptor activity and/or human VR3 receptor protein,human VR3 receptor DNA molecules including, but not limited to, thefollowing can be constructed:

Gene name Start codon End codon total base pairs VR3A+B+ 837 3065 2229VR3A+B− 338 2953 2616 VR3A−B−  1 2436 2436

(these numbers correspond to first nucleotide of first methionine andlast nucleotide before the first stop codon) and several constructscontaining portions of the CDNA encoding human VR3 receptor protein. Allconstructs can be designed to contain none, all or portions of the 5′ orthe 3′ untranslated region of human VR3 receptor cDNA. Human VR3receptor activity and levels of protein expression can be determinedfollowing the introduction, both singly and in combination, of theseconstructs into appropriate host cells. Following determination of thehuman VR3 receptor DNA cassette yielding optimal expression in transientassays, this Human VR3 receptor DNA construct is transferred to avariety of expression vectors, for expression in host cells including,but not limited to, mammalian cells, baculovirus-infected insect cells,E. coli, and the yeast S. cerevisiae.

Assay Methods for Human VR3 Receptor

Host cell transfectants and microinjected oocytes may be used to assayboth the levels of functional Human VR3 receptor activity and levels oftotal human VR3 receptor protein by the following methods. In the caseof recombinant host cells, this involves the co-transfection of one orpossibly two or more plasmids, containing the human VR3 receptor DNAencoding one or more fragments, subunits, or other functional gene. Inthe case of oocytes, this involves the co-injection of synthetic RNAsfor human VR3 receptor protein. Following an appropriate period of timeto allow for expression, cellular protein is metabolically labelledwith, for example ³⁵S-methionine for 24 hours, after which cell lysatesand cell culture supernatants are harvested and subjected toimmunoprecipitation with polyclonal antibodies directed against thehuman VR3 receptor protein.

Levels of human VR3 receptor protein in host cells are quantitated byimmunoaffinity and/or ligand affinity techniques. Human VR3receptor-specific affinity beads or human VR3 receptor-specificantibodies are used to isolate for example ³⁵S-methionine labelled orunlabelled human VR3 receptor protein. Labelled human VR3 receptorprotein is analyzed by SDS-PAGE. Unlabelled human VR3 receptor proteinis detected by Western blotting, ELISA or RIA assays employing human VR3receptor specific antibodies.

Other methods for detecting human VR3 receptor activity involve thedirect measurement of human VR3 receptor activity in whole cellstransfected with human VR3 receptor cDNA or oocytes injected with humanVR3 receptor mRNA. Human VR3 receptor activity is measured by specificligand binding or biological characteristics of the host cellsexpressing human VR3 receptor DNA. In the case of recombinant host cellsexpressing human VR3 receptor patch voltage clamp techniques can be usedto measure channel activity and quantitate human VR3 receptor protein.In the case of oocytes patch clamp as well as two-electrode voltageclamp techniques can be used to measure calcium channel activity andquantitate human VR3 receptor protein.

Cell Based Assays

The present invention provides a whole cell method to detect compoundmodulation of human VR3 receptor. The method comprises the steps;

1) contacting a compound, and a cell that contains functional human VR3receptor, and

2) measuring a change in the cell in response to modified human VR3receptor function by the compound.

The amount of time necessary for cellular contact with the compound isempirically determined, for example, by running a time course with aknown human VR3 receptor modulator and measuring cellular changes as afunction of time.

The measurement means of the method of the present invention can befurther defined by comparing a cell that has been exposed to a compoundto an identical cell that has not been similarly expose to the compound.Alternatively two cells, one containing functional human VR3 receptorand a second cell identical to the first, but lacking functional humanVR3 receptor could be both be contacted with the same compound andcompared for differences between the two cells. This technique is alsouseful in establishing the background noise of these assays. One ofaverage skill in the art will appreciate that these control mechanismsalso allow easy selection of cellular changes that are responsive tomodulation of functional human VR3 receptor.

The term “cell” refers to at least one cell, but includes a plurality ofcells appropriate for the sensitivity of the detection method. Cellssuitable for the present invention may be bacterial, yeast, oreukaryotic.

The assay methods to determine compound modulation of functional humanVR3 receptor can be in conventional laboratory format or adapted forhigh throughput. The term “high throughput” refers to an assay designthat allows easy analysis of multiple samples simultaneously, andcapacity for robotic manipulation. Another desired feature of highthroughput assays is an assay design that is optimized to reduce reagentusage, or minimize the number of manipulations in order to achieve theanalysis desired. Examples of assay formats include but are not limitedto, 96-well or 384-well plates, levitating droplets, and “lab on a chip”microchannel chips used for liquid handling experiments. It is wellknown by those in the art that as miniaturization of plastic molds andliquid handling devices are advanced, or as improved assay devices aredesigned, that greater numbers of samples may be performed using thedesign of the present invention.

The cellular changes suitable for the method of the present inventioncomprise directly measuring changes in the activity, function orquantity of human VR3 receptor, or by measuring downstream effects ofhuman VR3 receptor function, for example by measuring secondarymessenger concentrations or changes in transcription or by changes inprotein levels of genes that are transcriptionally influenced by humanVR3 receptor, or by measuring phenotypic changes in the cell. Preferredmeasurement means include changes in the quantity of human VR3 receptorprotein, changes in the functional activity of human VR3 receptor,changes in the quantity of mRNA, changes in intracellular protein,changes in cell surface protein, or secreted protein, or changes inCa+2, cAMP or GTP concentration. Changes in the quantity or functionalactivity of human VR3 receptor are described herein. Changes in thelevels of mRNA are detected by reverse transcription polymerase chainreaction (RT-PCR) or by differential gene expression. Immunoaffinity,ligand affinity, or enzymatic measurement quantitates VR3 inducedchanges in levels of specific proteins in host cells. Where the proteinis an enzyme, the induction of protein is monitored by cleavage of aflourogenic or colorimetric substrate.

Preferred detection means for cell surface protein include flowcytometry or statistical cell imaging. In both techniques the protein ofinterest is localized at the cell surface, labeled with a specificfluorescent probe, and detected via the degree of cellular fluorescence.In flow cytometry, the cells are analyzed in a solution, whereas incellular imaging techniques, a field of cells is compared for relativefluorescence.

The present invention is also directed to methods for screening forcompounds that modulate the expression of DNA or RNA encoding human VR3receptor as well as the function of human VR3 receptor protein in vivo.Compounds may modulate by increasing or attenuating the expression ofDNA or RNA encoding human VR3 receptor, or the function of human VR3receptor protein. Compounds that modulate the expression of DNA or RNAencoding human VR3 receptor or the function of human VR3 receptorprotein may be detected by a variety of assays. The assay may be asimple “yes/no” assay to determine whether there is a change inexpression or function. The assay may be made quantitative by comparingthe expression or function of a test sample with the levels ofexpression or function in a standard sample. Modulators identified inthis process are useful as therapeutic agents, and human VR3 receptor.

Purification of Human VR3 Receptor Protein

Following expression of human VR3 receptor in a recombinant host cell,human VR3 receptor protein may be recovered to provide purified humanVR3 receptor. Recombinant human VR3 receptor may be purified from celllysates and extracts, by various combinations of, or individualapplication of salt fractionation, ion exchange chromatography, sizeexclusion chromatography, hydroxylapatite adsorption chromatography andhydrophobic interaction chromatography, lectin chromatography, andantibody/ligand affinity chromatography.

Recombinant human VR3 receptor can be separated from other cellularproteins by use of an immunoaffinity column made with monoclonal orpolyclonal antibodies specific for full length nascent human VR3receptor, polypeptide fragments of human VR3 receptor or human VR3receptor subunits. The affinity resin is then equilibrated in a suitablebuffer, for example phosphate buffered saline (pH 7.3), and the cellculture supernatants or cell extracts containing human VR3 receptor orhuman VR3 receptor subunits are slowly passed through the column. Thecolumn is then washed with the buffer until the optical density (A₂₈₀)falls to background, then the protein is eluted by changing the buffercondition, such as by lowering the pH using a buffer such as 0.23 Mglycine-HC1 (pH 2.6). The purified Human VR3 receptor protein is thendialyzed against a suitable buffer such as phosphate buffered saline.

Production and Use of Antibodies that Bind to Human VR3 Receptor

Monospecific antibodies to human VR3 receptor are purified frommammalian antisera containing antibodies reactive against human VR3receptor or are prepared as monoclonal antibodies reactive with humanVR3 receptor using the technique originally described by Kohler andMilstein, Nature 256: 495-497 (1975). Immunological techniques are wellknown in the art and described in, for example, Antibodies: A laboratorymanual published by Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., ISBN 0879693142. Monospecific antibody as used herein isdefined as a single antibody species or multiple antibody species withhomogenous binding characteristics for human VR3 receptor. Homogenousbinding as used herein refers to the ability of the antibody species tobind to a specific antigen or epitope, such as those associated with thehuman VR3 receptor, as described above. Human VR3 receptor specificantibodies are raised by immunizing animals such as mice, rats, guineapigs, rabbits, goats, horses and the like, with rabbits being preferred,with an appropriate concentration of human VR3 receptor either with orwithout an immune adjuvant.

Preimmune serum is collected prior to the first immunization. Eachanimal receives between about 0.001 mg and about 100 mg of human VR3receptor associated with an acceptable immune adjuvant. Such acceptableadjuvants include, but are not limited to, Freund's complete, Freund'sincomplete, alum-precipitate, water in oil emulsion containingCorynebacterium parvum and tRNA. The initial immunization consists ofhuman VR3 receptor in, preferably, Freund's complete adjuvant atmultiple sites either subcutaneously (SC), intraperitoneally (IP) orboth. Each animal is bled at regular intervals, preferably weekly, todetermine antibody titer. The animals may or may not receive boosterinjections following the initial immunization. Those animals receivingbooster injections are generally given an equal amount of the antigen inFreund's incomplete adjuvant by the same route. Booster injections aregiven at about three-week intervals until maximal titers are obtained.At about 7 days after each booster immunization or about weekly after asingle immunization, the animals are bled, the serum collected, andaliquots are stored at about −20° C.

Monoclonal antibodies (mAb) reactive with human VR3 receptor areprepared by immunizing inbred mice, preferably Balb/c, with human VR3receptor. The mice are immunized by the IP or SC route with about 0.001mg to about 1.0 mg, preferably about 0.1 mg, of human VR3 receptor inabout 0.1 ml buffer or saline incorporated in an equal volume of anacceptable adjuvant, as discussed above. Freund's adjuvant is preferred,with Freund's complete adjuvant being used for the initial immunizationand Freund's incomplete adjuvant used thereafter. The mice receive aninitial immunization on day 0 and are rested for about 2 to about 30weeks. Immunized mice are given one or more booster immunizations ofabout 0.001 to about 1.0 mg of human VR3 receptor in a buffer solutionsuch as phosphate buffered saline by the intravenous (IV) route.Lymphocytes, from antibody positive mice, preferably spleniclymphocytes, are obtained by removing spleens from immunized mice bystandard procedures known in the art. Hybridoma cells are produced bymixing the splenic lymphocytes with an appropriate fusion partner,preferably myeloma cells, under conditions that will allow the formationof stable hybridomas. Fusion partners may include, but are not limitedto: mouse myelomas P3/NS1/Ag 4-1; MPC-11; S-194 and Sp2/0, with Sp2/0being generally preferred. The antibody producing cells and myelomacells are fused in polyethylene glycol, about 1000 mol. wt., atconcentrations from about 30% to about 50%. Fused hybridoma cells areselected by growth in hypoxanthine, thymidine and aminopterinsupplemented Dulbecco's Modified Eagles Medium (DMEM) by proceduresknown in the art. Supernatant fluids are collected from growth positivewells on about days 14, 18, and 21 and are screened for antibodyproduction by an immunoassay such as solid phase immunoradioassay(SPIRA) using human VR3 receptor as the antigen. The culture fluids arealso tested in the Ouchterlony precipitation assay to determine theisotype of the mAb. Hybridoma cells from antibody positive wells arecloned by a technique such as the soft agar technique of MacPherson,Soft Agar Techniques, in Tissue Culture Methods and Applications, Knuseand Paterson, Eds., Academic Press, 1973 or by the technique of limiteddilution.

Monoclonal antibodies are produced in vivo by injection of pristaneprimed Balb/c mice, approximately 0.5 ml per mouse, with about 1×10⁶ toabout 6×10⁶ hybridoma cells at least about 4 days after priming. Ascitesfluid is collected at approximately 8-12 days after cell transfer andthe monoclonal antibodies are purified by techniques known in the art.

In vitro production of anti-human VR3 receptor mAb is carried out bygrowing the hybridoma in tissue culture media well known in the art.High density in vitro cell culture may be conducted to produce largequantities of anti-human VR3 receptor MAbs using hollow fiber culturetechniques, air lift reactors, roller bottle, or spinner flasks culturetechniques well known in the art. The mAb are purified by techniquesknown in the art.

Antibody titers of ascites or hybridoma culture fluids are determined byvarious serological or immunological assays which include, but are notlimited to, precipitation, passive agglutination, enzyme-linkedimmunosorbent antibody (ELISA) technique and radioimmunoassay (RIA)techniques. Similar assays are used to detect the presence of human VR3receptor in body fluids or tissue and cell extracts.

It is readily apparent to those skilled in the art that the abovedescribed methods for producing monospecific antibodies may be utilizedto produce antibodies specific for human VR3 receptor polypeptidefragments, or full-length nascent human VR3 receptor polypeptide, or theindividual human VR3 receptor subunits. Specifically, it is readilyapparent to those skilled in the art that monospecific antibodies may begenerated which are specific for only one human VR3 receptor subunit orthe fully functional human VR3 receptor protein. It is also apparent tothose skilled in the art that monospecific antibodies may be generatedthat inhibit normal function of human VR3 receptor protein.

Human VR3 receptor antibody affinity columns are made by adding theantibodies to a gel support such that the antibodies form covalentlinkages with the gel bead support. Preferred covalent linkages are madethrough amine, aldehyde, or sulfhydryl residues contained on theantibody. Methods to generate aldehydes or free sulfhydryl groups onantibodies are well known in the art; amine groups are reactive with,for example, N-hydroxysuccinimide esters.

Kit Compositions Containing Human VR3 Receptor Specific Reagents

Kits containing human VR3 receptor DNA or RNA, antibodies to human VR3receptor, or human VR3 receptor protein may be prepared. Such kits areused to detect DNA that hybridizes to human VR3 receptor DNA or todetect the presence of human VR3 receptor protein or peptide fragmentsin a sample. Such characterization is useful for a variety of purposesincluding but not limited to forensic analyses, diagnostic applications,and epidemiological studies.

This invention relates to the use of human VR3 polynucleotides for theuse as diagnostic reagents. Detection of a mutated form of human VR3gene associated with a dysfunction will provide a diagnostic tool thatcan add to or define a diagnosis of a disease or susceptibility to adisease which results from under-expression or over-expression of humanVR3. Individuals carrying mutations in the human VR3 gene may bedetected at the DNA level by a variety of techniques well known in theare, and described herein.

The DNA molecules, RNA molecules, recombinant protein and antibodies ofthe present invention may be used to screen and measure levels of humanVR3 receptor DNA, human VR3 receptor RNA or human VR3 receptor protein.The recombinant proteins, DNA molecules, RNA molecules and antibodieslend themselves to the formulation of kits suitable for the detectionand typing of human VR3 receptor. Such a kit would comprise acompartmentalized carrier suitable to hold in close confinement at leastone container. The carrier would further comprise reagents such asrecombinant human VR3 receptor protein or anti-human VR3 receptorantibodies suitable for detecting human VR3 receptor. The carrier mayalso contain a means for detection such as labeled antigen or enzymesubstrates or the like.

Gene Therapy

Nucleotide sequences that are complementary to the human VR3 receptorencoding DNA sequence can be synthesized for antisense therapy. Theseantisense molecules may be DNA, stable derivatives of DNA such asphosphorothioates or methylphosphonates, RNA, stable derivatives of RNAsuch as 2′-O-alkylRNA, or other Human VR3 receptor antisenseoligonucleotide mimetics. Human VR3 receptor antisense molecules may beintroduced into cells by microinjection, liposome encapsulation or byexpression from vectors harboring the antisense sequence. Human VR3receptor antisense therapy may be particularly useful for the treatmentof diseases where it is beneficial to reduce human VR3 receptoractivity.

Human VR3 receptor gene therapy may be used to introduce human VR3receptor into the cells of target organisms. The human VR3 receptor genecan be ligated into viral vectors that mediate transfer of the human VR3receptor DNA by infection of recipient host cells. Suitable viralvectors include retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poliovirus and the like. Alternatively, human VR3receptor DNA can be transferred into cells for gene therapy by non-viraltechniques including receptor-mediated targeted DNA transfer usingligand-DNA conjugates or adenovirus-ligand-DNA conjugates, lipofectionmembrane fusion or direct microinjection. These procedures andvariations thereof are suitable for ex vivo as well as in vivo human VR3receptor gene therapy. Human VR3 receptor gene therapy may beparticularly useful for the treatment of diseases where it is beneficialto elevate human VR3 receptor activity. Protocols for molecularmethodology of gene therapy suitable for use with the human VR3 receptorgene is described in Gene Therapy Protocols, edited by Paul D. Robbins,Human press, Totawa N.J., 1996.

Pharmaceutical Compositions

Pharmaceutically useful compositions comprising human VR3 receptor DNA,human VR3 receptor RNA, or human VR3 receptor protein, or modulators ofhuman VR3 receptor activity, may be formulated according to knownmethods such as by the admixture of a pharmaceutically acceptablecarrier. Examples of such carriers and methods of formulation may befound in Remington's Pharmaceutical Sciences. To form a pharmaceuticallyacceptable composition suitable for effective administration, suchcompositions will contain an effective amount of the protein, DNA, RNA,or modulator.

Therapeutic or diagnostic compositions of the invention are administeredto an individual in amounts sufficient to treat or diagnose disorders inwhich modulation of Human VR3 receptor-related activity is indicated.The effective amount may vary according to a variety of factors such asthe individual's condition, weight, sex and age. Other factors includethe mode of administration. The pharmaceutical compositions may beprovided to the individual by a variety of routes such as subcutaneous,topical, oral and intramuscular.

The term “chemical derivative” describes a molecule that containsadditional chemical moieties that are not normally a part of the basemolecule. Such moieties may improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties mayattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Examples of such moieties are describedin a variety of texts, such as Remington's Pharmaceutical Sciences.

Compounds identified according to the methods disclosed herein may beused alone at appropriate dosages defined by routine testing in order toobtain optimal inhibition of the human VR3 receptor or its activitywhile minimizing any potential toxicity. In addition, co-administrationor sequential administration of other agents may be desirable.

The present invention also has the objective of providing suitabletopical, oral, systemic and parenteral pharmaceutical formulations foruse in the novel methods of treatment of the present invention. Thecompositions containing compounds or modulators identified according tothis invention as the active ingredient for use in the modulation ofhuman VR3 receptor can be administered in a wide variety of therapeuticdosage forms in conventional vehicles for administration. For example,the compounds or modulators can be administered in such oral dosageforms as tablets, capsules (each including timed release and sustainedrelease formulations), pills, powders, granules, elixirs, tinctures,solutions, suspensions, syrups and emulsions, or by injection. Likewise,they may also be administered in intravenous (both bolus and infusion),intraperitoneal, subcutaneous, topical with or without occlusion, orintramuscular form, all using forms well known to those of ordinaryskill in the pharmaceutical arts. An effective but non-toxic amount ofthe compound desired can be employed as a human VR3 receptor-modulatingagent.

The daily dosage of the products may be varied over a wide range from0.01 to 1,000 mg per patient, per day. For oral administration, thecompositions are preferably provided in the form of scored or unscoredtablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0,25.0, and 50.0 milligrams of the active ingredient for the symptomaticadjustment of the dosage to the patient to be treated. An effectiveamount of the drug is ordinarily supplied at a dosage level of fromabout 0.0001 mg/kg to about 100 mg/kg of body weight per day. The rangeis more particularly from about 0.001 mg/kg to 10 mg/kg of body weightper day. The dosages of the human VR3 receptor modulators are adjustedwhen combined to achieve desired effects. On the other hand, dosages ofthese various agents may be independently optimized and combined toachieve a synergistic result wherein the pathology is reduced more thanit would be if either agent were used alone.

Advantageously, compounds or modulators of the present invention may beadministered in a single daily dose, or the total daily dosage may beadministered in divided doses of two, three or four times daily.Furthermore, compounds or modulators for the present invention can beadministered in intranasal form via topical use of suitable intranasalvehicles, or via transdermal routes, using those forms of transdermalskin patches well known to those of ordinary skill in that art. To beadministered in the form of a transdermal delivery system, the dosageadministration will, of course, be continuous rather than intermittentthroughout the dosage regimen.

For combination treatment with more than one active agent, where theactive agents are in separate dosage formulations, the active agents canbe administered concurrently, or they each can be administered atseparately staggered times.

The dosage regimen utilizing the compounds or modulators of the presentinvention is selected in accordance with a variety of factors includingtype, species, age, weight, sex and medical condition of the patient;the severity of the condition to be treated; the route ofadministration; the renal and hepatic function of the patient; and theparticular compound thereof employed. A physician or veterinarian ofordinary skill can readily determine and prescribe the effective amountof the drug required to prevent, counter or arrest the progress of thecondition. Optimal precision in achieving concentrations of drug withinthe range that yields efficacy without toxicity requires a regimen basedon the kinetics of the drug's availability to target sites. Thisinvolves a consideration of the distribution, equilibrium, andelimination of a drug.

In the methods of the present invention, the compounds or modulatorsherein described in detail can form the active ingredient, and aretypically administered in admixture with suitable pharmaceuticaldiluents, excipients or carriers (collectively referred to herein as“carrier” materials) suitably selected with respect to the intended formof administration, that is, oral tablets, capsules, elixirs, syrups andthe like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Moreover, when desired or necessary,suitable binders, lubricants, disintegrating agents and coloring agentscan also be incorporated into the mixture. Suitable binders include,without limitation, starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include, without limitation, sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum and the like.

For liquid forms the active drug component can be combined in suitablyflavored suspending or dispersing agents such as the synthetic andnatural gums, for example, tragacanth, acacia, methyl-cellulose and thelike. Other dispersing agents that may be employed include glycerin andthe like. For parenteral administration, sterile suspensions andsolutions are desired. Isotonic preparations, which generally containsuitable preservatives, are employed when intravenous administration isdesired.

Topical preparations containing the active drug component can be admixedwith a variety of carrier materials well known in the art, such as,e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and Eoils, mineral oil, PPG2 myristyl propionate, and the like, to form,e.g., alcoholic solutions, topical cleansers, cleansing creams, skingels, skin lotions, and shampoos in cream or gel formulations.

The compounds or modulators of the present invention can also beadministered in the form of liposome delivery systems, such as smallunilamellar vesicles, large unilamellar vesicles and multilamellarvesicles. Liposomes can be formed from a variety of phospholipids, suchas cholesterol, stearylamine or phosphatidylcholines.

Compounds of the present invention may also be delivered by the use ofmonoclonal antibodies as individual carriers to which the compoundmolecules are coupled. The compounds or modulators of the presentinvention may also be coupled with soluble polymers as targetable drugcarriers. Such polymers can include polyvinyl-pyrrolidone, pyrancopolymer, polyhydroxypropylmethacrylamidephenol,polyhydroxy-ethylaspartamidephenol, or polyethyl-eneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds ormodulators of the present invention may be coupled to a class ofbiodegradable polymers useful in achieving controlled release of a drug,for example, polylactic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydro-pyrans,polycyanoacrylates and cross-linked or amphipathic block copolymers ofhydrogels.

For oral administration, the compounds or modulators may be administeredin capsule, tablet, or bolus form or alternatively they can be mixed inthe animals feed. The capsules, tablets, and boluses are comprised ofthe active ingredient in combination with an appropriate carrier vehiclesuch as starch, talc, magnesium stearate, or di-calcium phosphate. Theseunit dosage forms are prepared by intimately mixing the activeingredient with suitable finely-powdered inert ingredients includingdiluents, fillers, disintegrating agents, and/or binders such that auniform mixture is obtained. An inert ingredient is one that will notreact with the compounds or modulators and which is non-toxic to theanimal being treated. Suitable inert ingredients include starch,lactose, talc, magnesium stearate, vegetable gums and oils, and thelike. These formulations may contain a widely variable amount of theactive and inactive ingredients depending on numerous factors such asthe size and type of the animal species to be treated and the type andseverity of the infection. The active ingredient may also beadministered as an additive to the feed by simply mixing the compoundwith the feedstuff or by applying the compound to the surface of thefeed. Alternatively the active ingredient may be mixed with an inertcarrier and the resulting composition may then either be mixed with thefeed or fed directly to the animal. Suitable inert carriers include cornmeal, citrus meal, fermentation residues, soya grits, dried grains andthe like. The active ingredients are intimately mixed with these inertcarriers by grinding, stirring, milling, or tumbling such that the finalcomposition contains from 0.001 to 5% by weight of the activeingredient.

The compounds or modulators may alternatively be administeredparenterally via injection of a formulation consisting of the activeingredient dissolved in an inert liquid carrier. Injection may be eitherintramuscular, intraruminal, intratracheal, or subcutaneous. Theinjectable formulation consists of the active ingredient mixed with anappropriate inert liquid carrier. Acceptable liquid carriers include thevegetable oils such as peanut oil, cottonseed oil, sesame oil and thelike as well as organic solvents such as solketal, glycerol formal andthe like. As an alternative, aqueous parenteral formulations may also beused. The vegetable oils are the preferred liquid carriers. Theformulations are prepared by dissolving or suspending the activeingredient in the liquid carrier such that the final formulationcontains from 0.005 to 10% by weight of the active ingredient.

Topical application of the compounds or modulators is possible throughthe use of a liquid drench or a shampoo containing the instant compoundsor modulators as an aqueous solution or suspension. These formulationsgenerally contain a suspending agent such as bentonite and normally willalso contain an antifoaming agent. Formulations containing from 0.005 to10% by weight of the active ingredient are acceptable. Preferredformulations are those containing from 0.01 to 5% by weight of theinstant compounds or modulators.

The following examples illustrate the present invention without,however, limiting the same thereto.

EXAMPLE 1 Generation of Human Prostate and Pituitary cDNA Libraries cDNASynthesis

First Strand Synthesis

Approximately 5 μg of human prostate or pituitary mRNA (Clontech) wasused to synthesize cDNA using the cDNA synthesis kit (LifeTechnologies). Two microliters of Not1 primer adapter was added to 5 μlof mRNA and the mixture was heated to 70° C. for 10 minutes and placedon ice. The following reagents were added on ice: 41 μl of 5×firststrand buffer (250 mM TRIS-HCl (pH8.3), 375mM KCl, 15 mM MgCl₂), 2 μl of0.1M DTT, 10 mM dNTP (nucleotide triphosphates) mix and 1 μl of DEPCtreated water. The reaction was incubated at 42° C. for 5 minutes.Finally, 5 μl of Superscript RT II was added and incubated at 42° C. for2 more hours. The reaction was terminated on ice.

Second Strand Synthesis

The first strand product was adjusted to 93 μl with water and thefollowing reagents were added on ice: 30 μl of 5×2nd strand buffer (100mM TRIS-HCl (pH6.9), 450 mM KCl, 23 mM MgCl₂, 0.75 mM β-NAD+, 50 mM(NH₄)₂SO₄), 3 μl of 10 mM dNTP (nucleotide triphosphates), 1 μl E. coliDNA ligase (10 units) 1 μl RNase H (2 units), 4 μl DNA pol I (10 units).The reaction was incubated at 16° C. for 2 hours. The DNA from secondstrand synthesis was treated with T4 DNA polymerase and placed at 16° C.to blunt the DNA ends. The double stranded cDNA was extracted with 150μl of a mixture of phenol and chloroform (1:1, v:v) and precipitatedwith 0.5 volumes of 7.5 M NH4OAc and 2 volumes of absolute ethanol. Thepellet was washed with 70% ethanol and dried down at 37° C. to removethe residual ethanol. The double stranded DNA pellet was resuspended in25 μl of water and the following reagents were added; 10 μl of 5×T4 DNAligase buffer, 10 μl of Sal1 adapters and 5 μl of T4 DNA ligase. Theingredients were mixed gently and ligated overnight at 16° C. Theligation mix was extracted with phenol:chloroform:isoamyl alcohol,vortexed thoroughly and centrifuged at room temperature for 5 minutes at14,000×g to separate the phases. The aqueous phase was transferred to anew tube and the volume adjusted to 100 ml with water. The purified DNAwas size selected on a chromaspin 1000 column (Clontech) to eliminatethe smaller cDNA molecules. The double stranded DNA was digested withNot1 restriction enzyme for 3-4 hours at 37° C. The restriction digestwas electrophoresed on a 0.8% low melt agarose gel. The cDNA in therange of 1-5 kb was cut out and purified using Gelzyme (InVitrogen). Theproduct was extracted with phenol:chloroform and precipitated withNH₄OAc and absolute ethanol. The pellet was washed with 70% ethanol andresuspended in 10 ml of water.

Ligation of cDNA to the Vector

The cDNA was split up into 5 tubes (2μl each) and the ligation reactionswere set up by adding 4.5 μl of water, 2 pl of 5× ligation buffer, 1 μlof p-Sport vector DNA (cut with Sal-1/Not1 and phosphatase treated) and0.5 μl of T4 DNA ligase. The ligation was incubated at 40° C. overnight.

Introduction of Ligated cDNA into E. coli by Electroporation

The ligation reaction volume was adjusted to a total volume of 20 μlwith water. Five milliliters of yeast tRNA, 12.5 ml of 7.5M NH₄OAc and70 ml of absolute ethanol (−20° C.) was added. The mixture was vortexedthoroughly, and immediately centrifuged at room temperature for 20minutes at 14000×g. The pellets were washed in 70% ethanol and eachpellet was resuspended in 5 ml of water. All 5 ligations (25 ml) werepooled and 100μl of DH10B electro-competent cells (Life Technologies)were electroporated with 1 ml of DNA (total of 20 electroporations),then plated out on ampicillin plates to determine the number ofrecombinants (cfu) per microliter. The entire library was seeded into 2liters of Super Broth and maxipreps were made using Promega Maxi Prepkit and purified on cesium chloride gradients.

EXAMPLE 2 Library Screening/Human VR3 A+B+ Generation

Human Pituitary Gland Library Screening

One-microliter aliquots of the human pituitary gland-library wereelectroporated into Electromax DH10B cells (Life Technologies). Thevolume was adjusted to 1 ml with SOC media and incubated for 45 minutesat 37° C. with shaking. The library was then plated out on 150 cm²platescontaining LB to a density of 20000 colonies per plate. These cultureswere grown overnight at 37° C.

A human VR3 receptor probe was generated by polymerase chain reactionusing the following primer pair:

5′ oligo (SEQ.ID. NO.:1): 5′ ACCGGCCTATCCTCTTTGACATCGTG

3′ oligo (SEQ.ID.NO.:2): 5′ TGTCCGCCTTCTTGTGGGGGTTCTC

The probe was generated by PCR using regular PCR conditions using 5′ and3′ probe oligos (100 ng each) and 10 ng of diluted miniprep DNA. Theresulting 493 bp fragment was run on 1% agarose gel and purified using aQUIAquick Gel extraction kit (Quiagen). About 100 ng of the purifiedprobe was labeled with alpha 32P using oligolabeling kit from Pharmaciaand the labeled DNA was purified with S-200 columns (Pharmacia).

The library colonies were lifted on Protran nitrocellulose filters(Scheicher & Schuel) and the DNA was denatured in 1.5 M NaCl, 0.5 MNaOH. The filter disks were neutralized with 1.5 M NaCl, 1.0 M Tris-HCl,pH 7.5 and then UV crosslinked to the membrane using a UV-Stratalinker(Stratagene). The filters were washed several times in wash solution (1M Tris-HCl, pH 8.0; 5 M NaCl; 0.5 M EDTA; 20% SDS) at 42° C. Then thedisks were incubated in 1× southern pre-hybridization buffer (5′-3′ Inc)containing 50% formamide and 100 ug/ml of sheared salmon sperm DNA(5′-3′ Inc) for 6 hours at 42 C. Finally, hybridization was performedovernight at 42 C. in 1× hybridization buffer (5′-3′) containing 50%formamide, 100 ng of sheared salmon sperm DNA in the presence of labeledprobe (5×10⁵ to 1×10⁶ cmp/ml of hybridization buffer).

The disks were washed twice in 2×SSC, 0.2% SDS at room temperature (20minutes each) and once in 0.2×SSC, 0.1%SDS at 50 C. for 30 minutes. Themembranes were than placed on sheets of filter paper, wrapped in theplastic wrap and exposed to the film at −20 C. overnight.

Positive clones were identified and collected by coring the coloniesfrom the original plate. The colonies were incubated in LB for 1 hour at37° C. Dilutions of the cultures were plated onto LB agar plates and thefilter-lifting, hybridizing, washing, colony-picking procedure wasrepeated. Individual clones from the second screen were picked anddigested with SalI/NotI to determine the size of the inserts, and theinserts were sequenced.

The full length clone was generated by PCR with Pfu polymerase using 10ng of the sequenced library clone as a template and full length oligoswith KpnI (FL 5′ oligo SEQ.ID.NO.3) and NotI (FL 3′ oligo SEQ.ID.NO.4)sites.

FL 5′ oligo (SEQ.ID.NO.3):AACGTTGGTACCGCCACCATGGCGGATTCCAGCGAAGGCCCCCGCGCG

FL3′ oligo: (SEQ.ID.NO.4): TAAAGCGGCCGCTTCAGGAGGGACATCGGTGAGCCTCAC

The PCR product was digested with KpnI and NotI enzymes and cloned intoa pSP64T.GC expression vector. Large-scale preparation of DNA was doneusing a MEGA prep kit (Quiagen).

EXAMPLE 3 Library Screening/Human VR3 A+B− and Human VR3 A−B− Generation

Human Prostate Library Screening

One microliter aliquots of the human prostate library wereelectroporated into Electromax DH10B cells (Life Technologies). Thevolume was adjusted to 1 ml with SOC media and incubated for 45 minutesat 37° C. with shaking. The library was then plated out on 150 cm²plates containing LB to a density of 20000 colonies per plate. Thesecultures were grown overnight at 37° C.

A human VR3 receptor probe was generated by polymerase chain reactionusing the following primer pair:

5′ oligo (SEQ.ID. NO.:13): 5′ CTACCTGACGGAGAACCCCCACAAG

3′ oligo (SEQ.ID.NO.:14): 5′ GTAGTAGGCGGTGAGACTGAAGATGA

The probe was generated by PCR using regular PCR conditions using 5′ and3′ probe oligos (100 ng each) and 10 ng of diluted miniprep DNA. Theresulting 387 bp fragment was run on 1% agarose gel and purified using aQUIAquick Gel extraction kit (Quiagen). About 100 ng of the purifiedprobe was labeled with alpha 32P using oligolabeling kit from Pharmaciaand the labeled DNA was purified with S-200 columns (Pharmacia).

The library colonies were lifted on Protran nitrocellulose filters(Schleicher & Schuel) and the DNA was denatured in 1.5 M NaCl, 0.5 MNaOH. The filter disks were neutralized with 1.5 M NaCl, 1.0 M Tris-HCl,pH 7.5 and then UV crosslinked to the membrane using a UV-Stratalinker(Stratagene). The filters were washed several times in wash solution (1M Tris-HCl, pH 8.0; 5 M NaCl; 0.5 M EDTA; 20% SDS) at 42° C. Then thedisks were incubated in 1× southern pre-hybridization buffer (5′-3′ Inc)containing 50% formamide and 100 ug/ml of sheared salmon sperm DNA(5′-3′ Inc) for 6 hours at 42 C. Finally, hybridization was performedovernight at 42 C. in 1× hybridization buffer (5′-3′) containing 50%formamide, 100 ng of sheared salmon sperm DNA in the presence of labeledprobe (5×10⁵ to 1×10⁶ cmp/ml of hybridization buffer).

The disks were washed twice in 2×SSC, 0.2% SDS at room temperature (20minutes each) and once in 0.2×SSC, 0.1%SDS at 50 C. for 30 minutes. Themembranes were than placed on sheets of filter paper, wrapped in theSaran wrap and exposed to the film at −20 C. overnight.

Positive clones were identified and collected by coring the coloniesfrom the original plate. The colonies were incubated in LB for 1 hour at37° C. Dilutions of the cultures were plated onto LB agar plates and thefilter-lifting, hybridizing, washing, colony-picking procedure wasrepeated. Individual clones from the second screen were picked anddigested with EcoRI/NotI to determine the size of the inserts, and theinserts were sequenced.

The full length clone was generated by PCR with Pfu polymerase using 10ng of the sequenced library clone as a template and full length oligoswith NotI (FL 5′oligo SEQ.ID.NO.:15) and XbaI (FL 3′ oligoSEQ.ID.NO.:16) sites.

FL 5′ oligo (SEQ.ID.NO.:15):5′AACGTTGGCGGCCGCGCCACCATGGCGGATTCCAGCGAAGGCCCCCGCG CG

FL3′ oligo: (SEQ.ID.NO.:16): 5′ AACGTTTCTAGACTGGGCTGCAGTCCCTAG

The PCR product was digested with NotI and XbaI enzymes and cloned intoa pgem HE expression vector. Large-scale preparation of DNA was doneusing a MEGA prep kit (Quiagen).

EXAMPLE 4 Cloning Human VR3 Receptor cDNA into a Mammalian ExpressionVector

The human VR3 receptor cDNAs (collectively referred to as hVR3) werecloned into the mammalian expression vector pcDNA3.1/Zeo(+). The clonedPCR product was purified on a column (Wizard PCR DNA purification kitfrom Promega) and digested with Not I and EcoRI (NEB) to create cohesiveends. The product was purified by electrophoresis on a low melting pointagarose gel. The pcDNA3.1/Zeo(+) vector was digested with Not1 and XbaI(except for hVR3 A+B+, which was cloned into BamHI/NotI sites) enzymesand subsequently purified on a low melting point agarose gel. The linearvector was used to ligate to the human VR3 cDNA inserts. Recombinantswere isolated, designated human VR3 receptor, and used to transfectmammalian cells (HEK293, COS-7 or CHO-K1 cells) using the Effectenenon-liposomal lipid based transfection kit (Quiagen). Stable cell cloneswere selected by growth in the presence of zeocin. Single zeocinresistant clones were isolated and shown to contain the intact human VR3receptor gene. Clones containing the human VR3 receptor cDNAs wereanalyzed for hVR3 protein expression. Recombinant plasmids containinghuman VR3 encoding DNA were used to transform the mammalian COS or CHOcells or HEK293 cells.

Cells expressing human VR3 receptor, stably or transiently, are used totest for expression of human VR3 receptor and for ³H-RTX bindingactivity. These cells are used to identify and examine other compoundsfor their ability to modulate, inhibit or activate the human VR3receptor and to compete for radioactive ³H-RTX binding.

Cassettes containing the human VR3 receptor cDNA in the positiveorientation with respect to the promoter are ligated into appropriaterestriction sites 3′ of the promoter and identified by restriction sitemapping and/or sequencing. These cDNA expression vectors are introducedinto fibroblastic host cells for example COS-7 (ATCC# CRL1651), and CV-1tat [Sackevitz et al., Science 238: 1575 (1987)], 293, L (ATCC#CRL6362)] by standard methods including but not limited toelectroporation, or chemical procedures (cationic liposomes, DEAEdextran, calcium phosphate). Transfected cells and cell culturesupernatants are harvested and analyzed for human VR3 receptorexpression as described herein.

All of the vectors used for mammalian transient expression can be usedto establish stable cell lines expressing human VR3 receptor. UnalteredhumanVR3 receptor cDNA constructs cloned into expression vectors areexpected to program host cells to make human VR3 receptor protein. Thetransfection host cells include, but are not limited to, CV-1-P[Sackevitz et al., Science 238: 1575 (1987)], tk-L [Wigler, et al. Cell11: 223 (1977)], NS/0, and dHFr-CHO [Kaufman and Sharp, J. Mol. Biol.159: 601, (1982)].

Co-transfection of any vector containing human VR3 receptor cDNA with adrug selection plasmid including, but not limited to G418,aminoglycoside phosphotransferase; hygromycin, hygromycin-Bphosphotransferase; APRT, xanthine-guanine phosphoribosyl-transferase,will allow for the selection of stably transfected clones. Levels ofhuman VR3 receptor are quantitated by the assays described herein.

Human VR3 receptor cDNA constructs are also ligated into vectorscontaining amplifiable drug-resistance markers for the production ofmammalian cell clones synthesizing the highest possible levels of humanVR3 receptor. Following introduction of these constructs into cells,clones containing the plasmid are selected with the appropriate agent,and isolation of an over-expressing clone with a high copy number ofplasmids is accomplished by selection in increasing doses of the agent.

The expression of recombinant human VR3 receptor is achieved bytransfection of full-length human VR3 receptor cDNA into a mammalianhost cell.

EXAMPLE 5 Characterization of Functional Protein Encoded by pVR3R inXenopus oocytes

Xenopus laevis oocytes were prepared and injected using standard methodspreviously described and known in the art (Fraser et al., 1993). Ovarianlobes from adult female Xenopus laevis (Nasco, Fort Atkinson, Wis.) wereteased apart, rinsed several times in nominally Ca-free salinecontaining: 82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl₂, 5 mM HEPES, adjustedto pH 7.0 with NaOH (OR-2), and gently shaken in OR-2 containing 0.2%collagenase Type 1 (ICN Biomedicals, Aurora, Ohio) for 2-5 hours. Whenapproximately 50% of the follicular layers were removed, Stage V and VIoocytes were selected and rinsed in media consisting of 75% OR-2 and 25%ND-96. The ND-96 contained: 100 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mMCaCl₂, 5 mM HEPES, 2.5 mM Na pyruvate, gentamicin (50 ug/ml), adjustedto pH 7.0 with NaOH. The extracellular Ca⁺² was gradually increased andthe cells were maintained in ND-96 for 2-24 hours before injection. Forin vitro transcription, pGEM HE (Liman et al., 1992) containing humanVR3 was linearized with NheI and transcribed with T7 RNA polymerase(Promega) in the presence of the cap analog m7G(5′)ppp(5′)G. Thesynthesized cRNA was precipitated with ammonium acetate and isopropanol,and resuspended in 50 μl nuclease-free water. cRNA was quantified usingformaldehyde gels (1% agarose, 1×MOPS , 3% formaldehyde) against 1,2 and5 μl RNA markers (Gibco BRL, 0.24-9.5 Kb).

Oocytes were injected with 50 nl of the human VR3 receptor RNA (0.3-5ng) with or without co-injection of VR1 (2-3 ng). Control oocytes wereinjected with 50 nl of water. Oocytes were incubated for 1-13 days inND-96 before analysis for expression of the human VR3. Incubations andcollagenase digestion were carried out at room temperature. Injectedoocytes were maintained in 48 well cell culture clusters (Costar;Cambridge, Mass.) at 18° C. Whole cell agonist-induced currents weremeasured 1-14 days after injection with a conventional two-electrodevoltage clamp (GeneClamp500, Axon Instruments, Foster City, Calif.)using standard methods previously described and known in the art(Dascal, 1987). The microelectrodes were filled with 3 M KC1, which hadresistances of 1 and 2 MΩ. Cells were continuously perfused with ND96 at˜10 ml/min at room temperature unless indicated. Membrane voltage wasclamped at −70 mV unless indicated.

Oocytes were challenged with a variety of ligands, low pH, anddepolarizing as well as hyperpolarizing voltage steps but there were nodetectable differences in membrane conductance between humanVR3-expressing oocytes and control oocytes [see Table 1]. However, humanVR3 injected oocytes had properties that were different from controls in4 respects. First, injection of VR3 A+B− cRNA caused oocytes to die. Theviability of oocytes injected with VR3 isoforms having the A insert wassignificantly reduced compared to sister controls 3-4 days afterinjection (FIG. 9). Second, all three isoforms of VR3 enhanced theheat-induced response (FIG. 10A and 10B). Third, A+B− co-injection withhuman VR1 produced a ruthenium red sensitive perfusion-induced current(_(Iperfusion)) (FIGS. 11A and 11B). Fourth, human VR3 A+B− cRNAco-injection with human VR1 cRNA altered the responsiveness of theoocytes to 1 uM capsaicin, an agonist at the VR1 receptor (FIGS. 12A and12B).

TABLE 1 VR3 A + B − (3.25- 4 ng injected per oocyte VR3 A − B − (1.5 ngVR3 A + B + (5 ng Stimulus unless specified) injected per oocyte)injected per oocyte) Capsaicin (1 uM) NE: n = 3 (n = 30.4 ng) NE: n = 3NE: n = 3 Eugenol (10 uM) NE: n = 3 (n = 30.4 ng) NE: n = 3 NE: n = 3Olvanil (10 uM) NE: n = 3 (n = 10.4 ng) NE: n = 3 NE: n = 3Resiniferatoxin (1 uM) NE: n = 3 (n = 10.4 ng) NE: n = 3 NE: n = 3Piperine (10 uM) NE: n = 3 (n = 10.4 ng) NE: n = 3 NE: n = 3 Gingerol(10 uM) NE: n = 3 (n = 10.4 ng) NE: n = 3 NE: n = 3 Guiaicol (10 uM) NE:n = 3 (n = 10.4 ng) NE: n = 3 NE: n = 3 β-phenylethylamine (100 NE: n =2 NT NT uM) γ-hydroxybutyrate (100 NE: n = 2 NT NT uM) Anandamide (1.15uM; NE: n = 3 (0.4 ng NE: n = 1 NT RBI) injected) Arachadonic acid (10uM) NT NE: n = 3 NT pH 4.5-5.5 NE: n = 2 (4 ng cRNA); NE: n = 1 NE: n =1 n = 1 (0.4 ng cRNA) Depolarization No difference from No differencefrom No difference from control (n = 4) control (n = 6) control (n = 3)Hyperpolarization No difference from No difference from No differencefrom control (n = 4) control (n = 6) control (n = 3) NE: no effect; NT:not tested

Viability

Functional expression of human VR3 isoforms in Xenopus oocytes is shown:viability of oocytes maintained in ND-96 containing 2 Ca²⁺ wassignificantly diminished 4-6 days after injection with VR3 A+B− (3.25ng) and VR3 A+B+ (5 ng) but not VR3 A−B− (1.5 ng) cRNA. Data for VR3A+B− and control water-injected ootyes were similar in 5 separateexperiments and combined. Dead oocytes were determined visually using aBauch and Lomb dissecting microscope. Data were analyzed using Chisquare analysis. VR3 A+B− injected oocytes were the least viable: onlyabout 9% of oocytes survived to 4-6 days after injection. About 33% ofoocytes injected with human VR3 A+B+ survived to 4-6 days afterinjection. About 67% of sister water-injected control oocytes survivedthe same time period under essentially identical conditions. VR3 A−B−injected oocytes revealed similar viability to water injected controls.Similar data were obtained from 2 separate batches of cRNA.

Heat Responsiveness

Functional expression of human VR3 isoforms in Xenopus oocytes is shown:activation by heat. a). Shown is the mean peak current elicited by aheat ramp from 25 deg C. to 46 deg C. and maintained at 46 deg C. for atleast 15 sec. Voltage ramps were applied from −120 to +80 mV over 400msec at a sampling rate of 2 sec. The current shown was the increase incurrent over and above the initial current elicited at +80 mV (therightmost point on the current voltage curve shown in (b)). Oocytes wereinjected with 3.25, 1.5 and 5 ng VR3 A+B−, A−B−, and A+B+ cRNA,respectively, and recorded up to 6 days later. Solid bar: water-injectedcontrols (n=8); clear bar: VR3 A+B− isoform (n=1); hatched bar: VR3 A−B−isoform (n=8); stippled bar: VR3 A+B+ isoform (n=5). All isoforms showeda significant increase over water controls (p=0.001, 0.03 and 0.007,respectively). The heat-induced response was about 2-fold larger in theA+B− isoform compared to the other 2 isoforms, but the differences werenot significant (p=0.051 and p=0.16, for A+B− compared to A−B− and A+B+,respectively). Data were obtained from 2 sets of injected oocytes. Datashown is the mean and standard error of the mean. (b). Voltage-rampinduced currents were recorded during application of increasing heat tooocytes injected with water (top), VR3 A+B− (second from top), VR3 A−B−(3^(rd) from top), and VR3A+B+ (bottom). Oocytes were constantlyperfused with Ca2+ ND-96 and the solution was heated by an inline heaterdevice (TC-324B in conjunction with the SH-27A in line heater; WarnerInstrument Corp.). Ramp induced currents (in microAmperes, as indicatedon the y-axis) obtained at temperatures from 37 deg C. to 46 deg C. aredisplayed; only current traces at the higher temperatures are labeledwith the corresponding temperature.

Perfusion Induced Currents (I_(perfusion); FIGS. 11A and 14B)

The onset of bath perfusion elicited an increase in membrane conductancein oocytes that had been injected with RNA transcribed from the clonedhuman VR1 with and without RNA transcribed from VR3A+B− receptor cDNA asshown in FIGS. 11A AND 11B. An oocyte injected with VR1 cRNA (FIG. 11A)was challenged with voltage ramps between −120 and +80 mV over 400 msec.The ramp-induced currents were increased after onset of perfusion of theoocyte at a rate of 10 ml/min. Control ramp induced currents fromoocytes in still extracellular saline (C) were recorded prior to theonset of bath perfusion of Ca2+ ND-96. During perfusion, the rampinduced currents increased, indicating an increase in conductance (P).Subsequent perfusion of 10 uM ruthenium red (RR) did not block theperfusion induced current (P) in VR1-expressing oocytes. Thus, theperfusion-induced current observed in oocytes expressing VR1 alone wasinsensitive to 10 uM ruthenium red (A, “RR”). However, theperfusion-induced current elicited in oocytes expressing VR3A+B− and VR1were dramatically inhibited by 10 uM ruthenium red (B, “R”). Thisdifference was observed in 6 oocytes. The block was partially reversible(not shown). (FIG. 11B). An oocyte injected with VR1 cRNA together withVR3 A+B− was challenged with voltage ramps between −120 and +80 mV over400 msec. The ramp-induced currents were increased after onset ofperfusion of the oocyte at a rate of 10 ml/min. Perfusion activatedcurrents had similar magnitudes in both sets of oocytes. Vrev for the RRinhibited current was about −13 mV (arrow). The currents induced by bothagonists were strongly outwardly rectifying, as reported previously forthe rat VR1 (Caterina et al., 1997).

Capsaicin Induced Currents in Oocytes Expressing Human VR1 in thePresence or Absence of Human VR3 A+B− (FIGS. 12A and 12B)

Currents elicited by 1 uM capsaicin (FIGS. 12A and 12B) were measured ata holding voltage of +50 mV since responses were largest at this voltagedue to profound outward rectification of the capsaicin-induced currents(Caterina et al., 1997). Shown are 3 examples of oocytes injected withVR1 and water (a control for VR3 injection) (FIG. 12A) and oocytesinjected with VR1 cRNA and VR3 A+B− cRNA (FIG. 12B). Capsaicin was bathapplied during the time indicated by the solid horizontal bar. Themagnitude of the primary response to 1 uM capsaicin was diminished whenVR1 cRNA was co-expressed with VR3 A+B− cRNA at equal ratios (2.9 ngeach). The most notable difference was the magnitude and decay rate ofthe secondary response, the second hump that was usually obtained at thebeginning of the washout of capsaicin.

EXAMPLE 6 Characterization of Human VR3 in Mammalian Cell Lines

Human HEK293, CHO-K1 and COS-7 cells are transfected with human VR3isoforms pVR3A+B−R, pVR3A−B−R, or pVR3A+B+R,. Transient transfections 1μg of pVR3R per 10⁶ cells per 100 mm dish are performed using theEffectene tranfection kit (Quiagen; 301425). Three days aftertransfection, cells are plated onto 96-well plates (Biocoat,poly-D-lysine coated black/clear plate; Becton Dickinson part #354640).After one day, wells are rinsed with F12/DMEM, then incubated in Fluo-4(2 μM) with Pluronic F-127 (20%, 40 μl used in 20 mls total volume) for1 hour at room temperature. Plates are assayed using the FLIPR(Molecular Devices, FL-101).

Cells are challenged with solutions of different osmolarity (40 μl addedto 80 μl at a velocity of 50 μl/sec). Some wells are vigorously mixed tosimulate increased perfusion of the cells with extracellular solution.Transfections with vector alone are used as controls.

After three days the cells are selected in the presence of neomycin (200μg/ml) and grown through three 1:10 dilution passages for approximatelytwo weeks. Individual colonies are picked and grown in 6-well dishes.Cells are then plated onto 96-well plates (Biocoat, poly-D-lysine coatedblack/clear plate; Becton Dickinson part #354640) and grown toconfluence for three days. Wells are rinsed with F 12/DMEM, thenincubated in Fluo-4 (2 μM) with Pluronic acid (20%, 40 μl used in 20 mlstotal volume) for 1 hour at room temperature. Plates are assayed usingthe FLIPR (Molecular Devices, FL-101). Cells are challenged withagonists (at 3-fold concentration in 40 μl added to 80 μl at a velocityof 50 μl/sec).

The whole cell patch clamp technique (Hamill et al., 1981) is used torecord ligand-induced currents from HEK293 stably expressing human VR1receptor maintained for >2 days on 12 mm coverslips. Cells arevisualized using a Nikon Diaphot 300 with DIC Nomarski optics. Cells arecontinuously perfused in a physiological saline (˜0.5 ml/min) unlessotherwise indicated. The standard physiological saline (“Tyrodes”)contains: 130 mM NaCl, 4 mM KCl, 1 mM CaCl₂, 1.2 mM MgCl₂, and 10 mMhemi-Na-HEPES (pH 7.3, 295-300 mOsm as measured using a Wescor 5500vapor-pressure (Wescor, Inc., Logan, Utah). Recording electrodes arefabricated from borosilicate capillary tubing (R6; Garner Glass,Claremont, Calif.), the tips are coated with dental periphery wax (MilesLaboratories, South Bend, Ind.), and have resistances of 1-2 MΩ whencontaining intracellular saline: 100 mM K-gluconate, 25 mM KCl, 0.483 mMCaCl₂, 3 mM MgCl₂, 10 mM hemi-Na-HEPES and 1 mM K₄-BAPTA (100 nM freeCa⁺²); pH 7.4, with dextrose added to achieve 290 mOsm). Liquid junctionpotentials are −18 mV using standard pipette and bath solutions asdetermined both empirically and using the computer program JPCalc((Barry, 1994)). All voltages shown are corrected for liquid junctionpotential. Current and voltage signals are detected and filtered at 2kHz with an Axopatch ID patch-clamp amplifier (Axon Instruments, FosterCity, Calif.), digitally recorded with a DigiData 1200B laboratoryinterface (Axon Instruments), and PC compatible computer system andstored on magnetic disk for off-line analysis. Data acquisition andanalysis are performed with PClamp software. Slow changes in holdingcurrent are detected and filtered at 2 kHz, and recorded with a LPF202ADC amplifier (Warner, Hamden, Conn.) and VR10B digital data recorder(Instrutech, Great Neck, N.Y.) onto video tape. The signal is lateranalyzed at 10 Hz using Axotape software.

The total membrane capacitance (C_(m)) is determined as the differencebetween the maximum current after a 30 mV hyperpolarizing voltage rampfrom −68 mV generated at a rate of 10 mV/ms and the steady state currentat the final potential (−98 mV) (Dubin et al., 1999).

Apparent reversal potentials (V_(rev)) of ligand-induced conductancechanges are determined using a voltage-ramp protocol (Dubin et al.,1999). Voltage ramps are applied every 1 second and the resulting wholecell ramp-induced currents were recorded. Usually the voltage was rampedfrom negative to positive to negative values. The current required toclamp the cells at −68 mV is continuously monitored. Ligand-inducedconductances are determined from whole-cell currents elicited by avoltage-ramp protocol in the presence and absence of ligand. Voltageramp-induced currents measured before (control) and in the presence ofligand are compared to reveal the effect of the ligand on the channel tomodulate the channel current output. The voltage at which there is nonet ligand-induced current is determined (V_(rev)).

EXAMPLE 7 Primary Structure of the Human VR3 Receptor Protein

The present invention describes 3 isoforms of hVR3: A+B−, A−B−, andA+B+. The nucleotide sequences of human VR3 receptor cDNAs revealedsingle large open reading frame of about 2616, 2436 and 2229 base pairsencoding 871, 811, and 742 amino acids for human VR3 A+B−, A−B− andA+B+, respectively. The cDNA for VR3 A+B− has 5′ and 3′-untranslatedextensions of about 337 and about 547 nucleotides. The cDNA for VR3A+B+has 5′ and 3′-untranslated extensions of about 836 and about 994nucleotides. The first in-frame methionine was designated as theinitiation codon for an open reading frame that predicts human VR3receptor proteins with an estimated molecular mass (M_(r)) of about98,242 Da, 91,294 Da and 83,310 Da for the isoforms A+B−, A−B− and A+B+,respectively. The A+B− isoform encodes a protein of 871 amino acids. TheVR3 A−B− contains a deletion of 60 amino acids from amino acid 382 to441 of VR3A+B−. VR3 A+B+ is identical to A+B− until amino acid 736 afterwhich there are 6 divergent amino acids and a stop codon. The VR3 A+B+isoform extends 20 amino acids after the putative TM6.

The predicted human VR3 receptor proteins were aligned with nucleotideand protein databases and found to be related to the vanilloid receptorfamily (VR1 and VR2). There are several conserved motifs found in thisfamily of receptor including a large putative N-terminal hydrophilicsegment (about 467 amino acids), three putative ankyrin repeat domainsin the N-terminus region, 6 predicted transmembrane regions and a poreregion. VR3 A+B− is 43% identical to human VR1, 39% identical to bothhuman VRL-1 (AF129112) and human VRL (AF103906). Thus the VR3 receptordescribed herein is clearly a novel gene of the vanilloid receptorfamily.

VR3A+B− and VR3A−B− forms are very similar to the ratstretch-inhibitable channel SIC [genbank accession AB015231] from aminoacid 694 to the end. SIC, ecoded by 529 amino acids, is thought to forman ion channel inhibited by stretch. It lacks the large N-terminalcytoplasmic domain of the VR family but contains a sequence homologousto the A exon prior to the putative TM1.

The complete genomic sequence of the VR3 coding regions described hereinappears to be found in a 380512 base pair sequence submission to genbank(homo sapiens clone RPCI1-7G5 (AC007834), direct submission by Worley,K. C.). This genbank entry list many fragments of DNA sequence and aproposed contiguous sequence, but lacks any analysis of the nucleic acidsequence and fails to characterize the features of the VR3 nucleic acidsequences, or describe the presence of the VR3 gene.

1 Comparison of the sequences the present invention and the genomicsequence reveal that VR3 gene is composed of at least 15 exons. “A” is a60 amino acid insert in the putative N-terminal cytoplasmic domain thatfound in cDNAs obtained from both pituitary and prostate cDNA libraries.There are intron/exon border sequences at the A and B inserts.

The A+B− and A+B+ isoforms contain a domain in the N-terminal putativecytoplasmic region with homology to ankyrin repeat domain consensussequences. Thus, the VR3 A+ isoforms appear to have a similararchitecture as that predicted for VR1. The first domain wassignificantly similar to the consensus sequence (E=2.6 e-5). The next 2domains are not significant taken by themselves (e=37 and E=2.7) howevertaken as a whole, this region is likely to contain 3 ankyrin bindingdomains. The A− isoform is missing 20 amino acids of the putative 3^(rd)ankryn repeat domain and the juxtaposed sequence does not conform to anankyrin repeat domain.

The VR3 A+B− isoform has two potential myristoylation sites; [GPGGE] inN terminal and between TM4 and TM5. Putative phosphorylation sitesinclude: 7 potential PKC phosphorylation sites: T112, S134, T175, T190,T380, S403, S688 [however, S688 is in a putative extracellular region];1 potential PKA and PKG phosphorylation site: T181; 12 potential caseinkinase II phosphorylation sites: T89, S162, T181, T395, S416, S422,S432, T426, S432, S441, T740, S836; 3 potential mammary gland caseinkinase phosphorylation sites (S×E): S4, S726, S758; 1 potential tyrosinekinase phosphorylation site: Y411 [present in the “A” insert]; and 1potential N-linked glycosylation site: N651, [N201, N207 are in theputative intracellular N-terminal domain and are unlikely to beglycosylated]. There are no putative CaM binding domains in any isoformdescribed in the present invention.

In the A− isoform lacking the “A” insert 2 PKC phosphorylation sites, 6casein kinase II phosphorylation sites, and the only putative tyrosinekinase phosphorylation site is lacking.

The A+B+ isoform encodes a putative protein that lacks 2 casein kinaseII phosphorylation sites in the C-terminal putative intracellularregion.

EXAMPLE 8 Expression of Human VR3 in Tissues

Expression using a DNA array (Luo et al., 1999). DNA array distributionanalysis indicates that human VR3 mRNAs were expressed in a variety oftissues, and there was some overlap of expression with VR1 at a wholetissue level [FIG. 13]. The cRNA from the tissue type indicated in theleft column contained sequences encoding the human VR3 (middle column).Expression of human VR1 is shown in the right column and in most casesoverlapped with VR3 expression. NOTE: NS was non-significant expressionof human VR1. The sequence of human VR3 DNA immobilized on the DNA arraywas (SEQ.ID.NO.:17)

CCACCATCCTGGACATTGAGCGCTCCTTCCCCGTATTCCTGAGGAAGGCCTTCCGCTCTGGGGAGATGGTCACCGTGGGCAAGAGCTCGGACGGCACTCCTGACCGCAGTGGTGCTTCAGGGTGGATGAGGTGAACTGGTCTCACTGGAACCAGAACTTGGGCATCATCAACGAGGACCCGGGCAAGAATGAGACCTACC AGTATTATGGCTTCTCGCATACCGTGGGCCGCC.

This DNA sequence is not present in A+B+, only A+B− and A−B− isoforms.Note that the cDNA species was cloned from prostate gland (*; FIG. 13).

Northern blot analysis revealed expression of VR3 in whole brain,placenta, lung, kidney, pancreas and prostate.

EXAMPLE 9 Cloning Human VR3 Receptor cDNA into E. coli ExpressionVectors

Recombinant human VR3 receptor is produced in E. coli following thetransfer of the human VR3 receptor expression cassette into E. coliexpression vectors, including but not limited to, the pET series(Novagen). The pET vectors place human VR3 receptor expression undercontrol of the tightly regulated bacteriophage T7 promoter. Followingtransfer of this construct into an E. coli host that contain achromosomal copy of the T7 RNA polymerase gene driven by the induciblelac promoter, expression of human VR3 receptor is induced when anappropriate lac substrate (IPTG) is added to the culture. The levels ofexpressed human VR3 receptor are determined by the assays describedherein.

The cDNA encoding the entire open reading frame for human VR3 receptoris inserted into the NdeI site of pET [16] 11a. Constructs in thepositive orientation are identified by sequence analysis and used totransform the expression host strain BL21. Transformants are then usedto inoculate cultures for the production of human VR3 receptor protein.Cultures may be grown in M9 or ZB media, whose formulation is known tothose skilled in the art. After growth to an OD₆₀₀=1.5, expression ofhuman VR3 receptor is induced with 1 mM IPTG for 3 hours at 37° C.

EXAMPLE 10 Cloning Human VR3 Receptor cDNA into a Baculovirus ExpressionVector for Expression in Insect Cells

Baculovirus vectors, which are derived from the genome of the AcNPVvirus, are designed to provide high level expression of cDNA in the Sf9line of insect cells (ATCC CRL#1711). Recombinant baculovirusesexpressing human VR3 receptor cDNA is produced by the following standardmethods (In Vitrogen Maxbac Manual): the human VR3 receptor cDNAconstructs are ligated into the polyhedrin gene in a variety ofbaculovirus transfer vectors, including the pAC360 and the BlueBacvector (In Vitrogen). Recombinant baculoviruses are generated byhomologous recombination following co-transfection of the baculovirustransfer vector and linearized AcNPV genomic DNA [Kitts, P. A., Nuc.Acid. Res. 18: 5667 (1990)] into Sf9 cells. Recombinant pAC360 virusesare identified by the absence of inclusion bodies in infected cells andrecombinant pBlueBac viruses are identified on the basis ofβ-galactosidase expression (Summers, M. D. and Smith, G. E., TexasAgriculture Exp. Station Bulletin No. 1555). Following plaquepurification, human VR3 receptor expression is measured by the assaysdescribed herein.

The cDNA encoding the entire open reading frame for human VR3 receptoris inserted into the BamItI site of pBlueBacII. Constructs in thepositive orientation are identified by sequence analysis and used totransfect Sf9 cells in the presence of linear AcNPV mild type DNA.

Authentic, active human VR3 receptor is found in the cytoplasm ofinfected cells. Active human VR3 receptor is extracted from infectedcells by hypotonic or detergent lysis.

EXAMPLE 11 Cloning Human VR3 Receptor cDNA into a Yeast ExpressionVector

Recombinant human VR3 receptor is produced in the yeast S. cerevisiaefollowing insertion of the optimal human VR3 receptor cDNA cistron intoexpression vectors designed to direct the intracellular or extracellularexpression of heterologous proteins. In the case of intracellularexpression, vectors such as EmBLyex4 or the like are ligated to thehuman VR3 receptor cistron [Rinas, U. et al., Biotechnology 8: 543-545(1990); Horowitz B. et al., J. Biol. Chem. 265: 4189-4192 (1989)]. Forextracellular expression, the human VR3 receptor cistron is ligated intoyeast expression vectors which fuse a secretion signal (a yeast ormammalian peptide) to the NH₂ terminus of the human VR3 receptor protein[Jacobson, M. A., Gene 85: 511-516 (1989); Riett L. and Bellon N.Biochem. 28: 2941-2949 (1989)].

These vectors include, but are not limited to pAVE 1>6, which fuses thehuman serum albumin signal to the expressed cDNA [Steep O. Biotechnology8: 42-46 (1990)], and the vector pL8PL which fuses the human lysozymesignal to the expressed cDNA [Yamamoto, Y., Biochem. 28: 2728-2732)]. Inaddition, human VR3 receptor is expressed in yeast as a fusion proteinconjugated to ubiquitin utilizing the vector pVEP [Ecker, D. J., J.Biol. Chem. 264: 7715-7719 (1989), Sabin, E. A., Biotechnology 7:705-709 (1989), McDonnell D. P., Mol. Cell Biol. 9: 5517-5523 (1989)].The levels of expressed human VR3 receptor are determined by the assaysdescribed herein.

EXAMPLE 12 Purification of Recombinant Human VR3 Receptor

Recombinantly produced human VR3 receptor may be purified by antibodyaffinity chromatography.

Human VR3 receptor antibody affinity columns are made by adding theanti-human VR3 receptor antibodies to Affigel-10 (Bio-Rad), a gelsupport that is pre-activated with N-hydroxysuccinimide esters such thatthe antibodies form covalent linkages with the agarose gel bead support.The antibodies are then coupled to the gel via amide bonds with thespacer arm. The remaining activated esters are then quenched with 1Methanolamine HCl (pH 8). The column is washed with water followed by0.23 M glycine HCl (pH 2.6) to remove any non-conjugated antibody orextraneous protein. The column is then equilibrated in phosphatebuffered saline (pH 7.3) together with appropriate membrane solubilizingagents such as detergents and the cell culture supernatant or cellextract containing solubilized human VR3 receptor is slowly passedthrough the column. The column is then washed with phosphate-bufferedsaline together with detergents until the optical density (A280) fallsto background, then the protein is eluted with 0.23 M glycine-HCl (pH2.6) together with detergents. The purified human VR3 receptor proteinis then dialyzed against phosphate buffered saline.

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What is claimed is:
 1. An isolated and purified nucleic acid moleculewhich encodes a human Vanilloid Receptor 3 (VR3) protein, wherein saidprotein comprises the amino acid sequence set forth in SEQ ID NO:
 7. 2.The isolated and purified nucleic acid molecule of claim 1, having anucleotide sequence selected from a group consisting of SEQ ID NO: 5 andSEQ ID NO:
 6. 3. The isolated and purified nucleic acid molecule ofclaim 1, wherein said molecule is selected from a group consisting ofDNA, RNA, and cDNA.
 4. An expression vector comprising a nucleic acidsequence encoding human Vanilloid Receptor 3 (VR3) protein, wherein saidprotein comprises the amino acid sequence set forth in SEQ ID NO:
 7. 5.The expression vector of claim 4, wherein the nucleic acid sequenceencoding human VR3 protein is selected from a group consisting of SEQ IDNO: 5 and SEQ ID NO:
 6. 6. The expression vector of claim 4, wherein theexpression vector contains DNA encoding human VR3 protein.
 7. A processfor expression of human Vanilloid Receptor 3 (VR3) protein in arecombinant host cell comprising: a) transferring the expression vectorof claim 4 into suitable host cells; and b) culturing the host cells ofstep (a) under conditions which allow expression of the human VR3protein from the expression vector.
 8. The recombinant host cell ofclaim 7, wherein said expression vector comprises a nucleotide sequenceselected from a group consisting of SEQ ID NO: 5 and SEQ ID NO:
 6. 9. Aprocess for expression of human Vanilloid Receptor 3 (VR3) protein in arecombinant host cell comprising: transferring the expression vector ofclaim 4 into suitable host cells; and culturing the host cells of step(a) under conditions which allow expression of the human VR3 proteinfrom the expression vector.